Pseudomonas aeruqinosa is a gram negative bacterium which primarily causes infection in compromised patients such as those with cancer, burns or Cystic Fibrosis. In comparison to other gram negative infections these infections have a particularly high mortality rate. This organism produces a varitey of extracellular factors which may play a significant role in the pathogenesis of human infections, but the actual function of these determinants in even one kind of Pseudomonas infection is not yet fully understood. Additionally, it has become clear that the virulence of Pseudomonas is dependent upon the ability of this organism to scavenge certain nutrients which are limiting during an infection, such as iron and inorganic phosphate. Interestingly, these nutrients also regulate the expression of virulence factors (toxins) of Pseudomonas. Therefore, an understanding of the regulation of these toxins is critical to understanding the pathogenesis of Pseudomonas. The overall goal of this research is to understand the role of Exotoxin A (ETA) and phospholipase C (PLC) in the pathogenesis of P. aeruginosa and to elucidate how the regulation of these toxins contributes to the virulence of this opportunistic pathogen. These studies may lead to the rational development of improved therapeutic and prophylactic measures for infections caused by this organism. The immediate objectives are to use biochemical, immunological and recombinant DNA methods to characterize the structural genes encoding these toxins and to determine at the molecular level the mechanisms regulating their synthesis. Gene fusion methods will also be used to study the regulation of ETA and PLC synthesis. We will use recombinant DNA methods and classical microbial genetics to construct precisely defined mutants which are altered in their ability to produce ETA and PLC. These mutants will then be characterized in biological models (cell culture and whole animal) relevant to Pseudomonas infections.